The cholesterol-dependent cytolysins (CDCs) are a family of bacterial toxins that are important virulence factors for a number of pathogenic Gram-positive bacterial species. CDCs are secreted as soluble, stable monomeric proteins that bind specifically to cholesterol-rich cell membranes, where they assemble into well-defined ring-shaped complexes of around 40 monomers. The complex then undergoes a concerted structural change, driving a large pore through the membrane, potentially lysing the target cell. Understanding the details of this process as the protein transitions from a discrete monomer to a complex, membrane-spanning protein machine is an ongoing challenge. While many of the details have been revealed, there are still questions that remain unanswered. In this review, we present an overview of some of the key features of the structure and function of the CDCs, including the structure of the secreted monomers, the process of interaction with target membranes, and the transition from bound monomers to complete pores. Future directions in CDC research and the potential of CDCs as research tools will also be discussed.
The cholesterol-dependent cytolysins (CDCs) are bacterial, β-barrel, pore-forming toxins. A central enigma of the pore-forming mechanism is how completion of the prepore is sensed to initiate its conversion to the pore. We identified a motif that is conserved between the CDCs and a diverse family of nearly 300 uncharacterized proteins present in over 220 species that span at least 10 bacterial and 2 eukaryotic phyla. Except for this motif, these proteins exhibit little similarity to the CDCs at the primary structure level. Studies herein show this motif is a critical component of the sensor that initiates the prepore-to-pore transition in the CDCs. We further show by crystallography, single particle analysis, and biochemical studies of one of these CDC-like (CDCL) proteins from Elizabethkingia anophelis, a commensal of the malarial mosquito midgut, that a high degree of structural similarity exists between the CDC and CDCL monomer structures and both form large oligomeric pore complexes. Furthermore, the conserved motif in the E. anophelis CDCL crystal structure occupies a nearly identical position and makes similar contacts to those observed in the structure of the archetype CDC, perfringolysin O (PFO). This suggests a common function in the CDCs and CDCLs and may explain why only this motif is conserved in the CDCLs. Hence, these studies identify a critical component of the sensor involved in initiating the prepore-to-pore transition in the CDCs, which is conserved in a large and diverse group of distant relatives of the CDCs. IMPORTANCE The cholesterol-dependent cytolysins’ pore-forming mechanism relies on the ability to sense the completion of the oligomeric prepore structure and initiate the insertion of the β-barrel pore from the assembled prepore structure. These studies show that a conserved motif is an important component of the sensor that triggers the prepore-to-pore transition and that it is conserved in a large family of previously unidentified CDC-like proteins, the genes for which are present in a vast array of microbial species that span most terrestrial environments, as well as most animal and human microbiomes. These studies establish the foundation for future investigations that will probe the contribution of this large family of CDC-like proteins to microbial survival and human disease.
The cholesterol-dependent cytolysins (CDCs) are a major family of bacterial pore-forming proteins secreted as virulence factors by Gram-positive bacterial species. CDCs are produced as soluble, monomeric proteins that bind specifically to cholesterol-rich membranes, where they oligomerize into ring-shaped pores of more than 30 monomers. Understanding the details of the steps the toxin undergoes in converting from monomer to a membrane-spanning pore is a continuing challenge. In this review we summarize what we know about CDCs and highlight the remaining outstanding questions that require answers to obtain a complete picture of how these toxins kill cells. K E Y W O R D S CD59, cholesterol-binding protein, cholesterol-dependent cytolysin, intermedilysin, MACPF, membrane-protein interactions, perfringolysin O, pneumolysin, pore-forming toxin 1 | INTRODUCTION Pore-forming toxins (PFTs), bacterial toxins that act via membrane permeabilization, are typically produced to aid bacterial virulence or survival. Cholesterol-dependent cytolysins (CDCs) are a large family of PFTs observed in numerous Gram-positive bacterial genera including Streptococcus, Clostridium, Listeria, and Gardnerella.
The spike (S) glycoprotein of SARS CoV-2 is the target of neutralizing antibodies (NAbs) that are crucial for vaccine effectiveness. The S1 subunit binds ACE2 while the S2 subunit mediates virus-cell membrane fusion. S2 is a class I fusion glycoprotein subunit and contains a central coiled coil that acts as a scaffold for the conformational changes associated with fusion function. The coiled coil of S2 is unusual in that the 3–4 repeat of inward-facing positions are mostly occupied by polar residues that mediate few inter-helical contacts in the prefusion trimer. We examined how insertion of bulkier hydrophobic residues (Val, Leu, Ile, Phe) to fill a cavity next to Ala1016 and Ala1020 in the 3–4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala1016 with bulkier hydrophobic residues in the context of a prefusion-stabilized S trimer, S2P-FHA, was associated with increased thermal stability. S glycoprotein membrane fusion function was retained with Ala1016/Ala1020 cavity-filling mutations associated with improved recombinant S2P-FHA thermostability, however 2 mutants, A1016L and A1016V/A1020I, lacked ability to mediate entry of S-HIV-1 pseudoparticles into 293-ACE2 cells. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited neutralizing antibody with 50%-inhibitory dilutions (ID50s) in the range 2,700–5,110 for ancestral and Delta-derived viruses, and 210–1,744 for Omicron BA.1. The antigens elicited antibody specificities directed to the receptor-binding domain (RBD), N-terminal domain (NTD), fusion peptide and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S2P-FHA-like ectodomain oligomers in the absence of an external trimerization motif (T4 foldon), thus representing an alternative approach for stabilizing oligomeric S glycoprotein vaccines.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.